Antibiotic TKR2999, process for the preparation thereof and microbe

ABSTRACT

The antibiotic TKR2999 having the physicochemical properties described below and its pharmacologically acceptable salt: 
     (1) FAB-MS m/z 971 [M+H)] + , (2) the molecular formula: C 44 H 78 N 10 O 14 , and high-resolution FAB-MS m/z 971.5776 [M+H] + , (3) the ultraviolet absorption spectrum in methanol has an end absorption, (4) the infrared absorption spectrum by KBr method shows the major absorption wave numbers at 3320, 2920, 1680, 1540, 1210, 1140, 840, 800, and 720 cm −1 , (5) aspartic acid, threonine, serine, glycine, alanine, β-alanine, and ornithine are detected by the amino acid analysis using ninhydrin reaction, and (6) the solubility is that it is soluble in methanol, and practically insoluble in hexane, chloroform, and water.

TECHNICAL FIELD

The present invention relates to the antibiotic TKR2999, which is usefulas a therapeutic agent for fungal infection diseases, a method for itsproduction, and microorganisms producing it.

BACKGROUND ART

Fungi are known to cause a variety of infectious diseases in man,animals, and plants. In man, for instance, they cause superficialmycosis affecting the skin, oral cavity, etc. and systemic mycosisaffecting the viscera, brain, etc. They cause similar infections in petand domestic animals as well. Furthermore, fungi inflict varioushazardous effects on plants such as orchard trees and vegetables.

As the principal pathogenic fungi causing systemic mycosis in man, thoseof the genera Candida, Cryptococcus, and Aspergillus, among others, areknown. As to superficial mycosis, Candida species affecting the skin,oral cavity, and vagina and trichophytons infecting the skin of theextremities are regarded as the major pathogenic fungi. Besides thosefungi, many other fungi exist in the environment and are suspected tocontaminate the animal and vegetable kingdoms.

As antimycotics of use for the prevention and treatment of such fungalinfections and contaminations, only a very few are known. As therapeuticdrugs for systemic mycosis in man and animals, for instance,amphotericin B, flucytosine, miconazole, and fluconazole can bementioned. However, those compounds are not fully satisfactory inpotency, toxic potential, or antifungal spectrum, thus being notimpeccable as therapeutic drugs.

In view of the above-mentioned prior art, the present invention has forits object to provide a novel antibiotic which is of value as atherapeutic agent for fungal infections.

In the search for a novel antibiotic, the inventors of the presentinvention isolated a large number of microorganisms from the naturalkingdom, isolated the antibiotics they produced, and scrutinized theirbiological properties. As a result, they discovered that the culturebroth of a strain of microorganism of the Fungi Imperfecti contained anantibiotic having antifungal activity against pathogenic fungi inclusiveof Candida albicans, C. kefyr, Cryptococcus neoformans, and Aspergillusfumigatus. Accordingly the inventors isolated this antibiotic andstudied its physicochemical properties. As a result, they discoveredthat it was a novel substance having distinct physicochemicalcharacteristics, which had been described in no literatures yet, andnamed it TKR2999. The present invention intends to provide the aboveantibiotic TKR2999 and a method of production thereof.

The present inventions are described in detail as follows.

DISCLOSURE OF THE INVENTION

The above antibiotic TKR2999 has the physicochemical properties of (1),(2), (3), (4), and (5) described below:

(1) the mass spectrumby FAB-MS method gives an ion peak of [M+H]⁺ at m/z971;

(2) the molecular formula is represented by C₄₄H₇₈N₁₀O₁₄, and its massspectrum by high-resolution FAB-MS gives m/z 971.5776 as [M+H]⁺;

(3) the ultraviolet absorption spectrum in methanol has an endabsorption;

(4) the infrared absorption spectrum by KBr method shows the majorabsorption wave numbers at 3320, 2920, 1680, 1540, 1210, 1140, 840, 800,and 720 cm⁻¹;

(5) aspartic acid, threonine, serine, glycine, alanine, β-alanine, andornithine are detected by the amino acid analysis using ninhydrinreaction; and

(6) the solubility is that it is soluble in methanol, and practicallyinsoluble in hexane, chloroform, and water.

The antibiotic TKR2999 mentioned above shows the ¹H-NMR spectrum shownin FIG. 3 and the ¹³C-NMR spectrum shown in FIG. 4, and is characterizedin the reversed-phase high-performance liquid chromatography, in whichit is eluted at the position indicated in FIG. 5.

The above-mentioned antibiotic TKR2999 can be produced by growing astrain of microorganism belonging to the class Fungi Imperfecti andcapable of producing said TKR2999 in a culture medium, and by isolatingthe substance from the culture broth of the above strain.

The strain of microorganism used in the present invention is not limitedand can be used only provided it is capable of producing said TKR2999.An example of the strain that is used for production of the saidcompound is the fungi TKR2999 (hereinafter referred to as theTKR2999-strain) belonging to the class of Fungi Imperfecti.

The above-mentioned TKR2999-strain is a novel strain not heretoforedescribed in a literature, and was isolated and characterized for thefirst time by the inventors of the present invention. The strain has theproperty to produce TKR2999 with advantage. The mycologicalcharacteristics of this TKR2999-strain are now described in detail asfollows.

The colors of colonies of said TKR2999-strain on various media are shownin Table 1. The description of colors in the table is based on thoseprescribed in Japanese Industrial Standard (JIS) Z 8102 (1985). Theresults of observation on days 14 of culture at 25° C. after inoculationare shown.

TABLE 1 Diameter Surface of colony Color of color of Appearance Medium(mm) colony colony of colony Malt 29 grayish sepia velvety extractyellow-red 10YR3/2 agar 5YR5/2 Potato 37 dark gray little dark velvetydextrose N3 yellow agar 2.5Y3/2 Sabouraud 28 dark yellowish little darkvelvety agar gray yellow 5Y4/1 2.5Y3/2 YpSs agar 26 light dark grayishvelvety yellowish gray blue-green 5YR7/1 2.5BG3/2

The above TKR2999-strain grows slowly on malt extract agar, potatodextrose agar, and Sabouraud agar etc. The colony has velvety surfaceand rises in the center with dense hard mycelia. Conidia of theTKR2999-strain are unicellular, cylindrical with smooth surface, and theboth edges are round. Their size is 3-8×1.5-2.5 μm. Conidia are formedwell on the above medium, but the conidia formation style in view of theconidiophore is uncharacterized.

Among the mycological characters of the TKR2999-strain, itsphysiological characteristics are as follows.

Temperature range for growth: the temperature range for growth is 10 to30° C. and the optimum range of temperature for growth is around 25° C.

The pH range for growth: the pH range for growth is pH 3 to 9 and theoptimum range of pH for growth is pH 5.

The above mycological characters are compared with the descriptions ofspecies of the class Fungi Imperfecti described in “A Manual of SoilFungi” authored by Joseph C. Gilman (Constable and company Ltd.) (1959)etc. The species of the TKR2999-strain can not be identified because itsconidia formation style is unidentified.

However, no report was available on a strain of microorganism having theability to product TKR2999 among strains of the class Fungi Imperfecti.Therefore, the inventors of the present invention regarded it as a novelstrain and named it Fungi strain TKR2999 of the Fungi Imperfecti. Thestrain was deposited under the Budapest Treaty with the NationalInstitute of Bioscience and Human Technology (Address, 1-3, Higashi1-chome, Tsukuba-shi, Tbaraki, Japan (Zip code 305-0046)) under theaccession number of FERM BP-6524 (original date of deposit: Nov. 21,1997; date of request for transfer to international deposit: Sep. 24,1998).

The present invention can be carried into practice not only with theabove-mentioned TKR2999-strain but also with any spontaneous orartificial mutant of the TKR2999-strain or any other strain ofmicroorganism belonging to the class Fungi Imperfecti and capable ofproducing TKR2999.

In accordance with the present invention, TKR2999 is produced bycultivating a TKR2999-producing strain described above in a nutrientmedium. Nutrients to be used for the medium include various carbonsources such as glucose, fructose, saccharose, starch, dextrin,glycerol, molasses, malt syrup, oils and fats, and organic acids.

Nutrients to be used for the medium include nitrogen sources, organicand inoragnic materials such as soybean meal, cotton seed meal, cornsteep liquor, casein, peptone, yeast extract, meat extract, wheat germs,urea, amino acids, ammonium salts, etc. Salts as nutrients are variousinorganic salts such as salts of sodium, potassium, calcium, magnesium,etc. and salts of phosphoric acid. Those materials can be usedindependently or in a suitable combination.

Where necessary, the nutrient medium may be supplemented with heavymetal salts such as iron salts, copper salts, zinc salts, cobalt salts,etc., vitamins such as biotin, vitamin B1, etc., and other organic andinorganic substances which would assist in growth of the microorganismand promote production of TKR2999.

In addition to the above components, an antifoamer and/or a surfactant,for example silicone oil, polyalkylene glycol ethers, etc., can be addedto the nutrient medium.

In cultivating a strain of microorganism capable of producing TKR2999 insaid nutrient medium, a variety of cultural methods which are generallyused in the production of antibiotics by means of microorganisms can beemployed. However, a liquid culture method, particularly a method byshake culture or submerged aerobic culture, is preferred.

The cultivation is preferably carried out at 15 to 25° C. The pH of themedium may range from pH 3 to 8 and is preferably around pH 5. Regardingthe incubation time, generally a sufficient output of the substance canbe expected by 6 to 15 days of culture.

By means of the above cultivation, TKR2999 is contained bothintracellularly and extracellularly and accumulated in the culturebroth. In the present invention, the TKR2999 accumulated in the culturebroth can be recovered and isolated from the broth by utilizing itsphysicochemical characteristics and, where necessary, by furtherpurification.

The above-mentioned recovery can be achieved by extracting the wholebroth with a non-hydrophilic organic solvent such as ethyl acetate,butyl acetate, chloroform, butanol, methyl isobutyl ketone, or the like.As an alternative, it is possible to subject the broth to centrifugationor filtration to separate into the medium and cells and isolate theantibiotics from each of the medium and cells.

For separation of TKR2999 from the medium not only the above-mentionedextraction method using a non-hydrophilic organic solvent but also themethod which comprises contacting the medium with an adsorbent to letTKR2999 adsorbed on the adsorbent and desorbing or eluting them with asolvent can be employed.

The adsorbent that can be used includes, for example, activated carbon,cellulose powder, and adsorbent resins. As the above-mentioned solvent,a variety of solvents can be selectively used according to the kind andproperties of the adsorbent and either singly or in combination. Thus,an aqueous solution of one or more water-soluble organic solvents, suchas aqueous acetone, aqueous alcohol, etc., can be employed. Forseparation of TKR2999 from the cells, the extraction technique using ahydrophilic organic solvent such as acetone can be employed.

In the present invention, the crude extract of TKR2999 obtained from theculture broth as described above can be subjected to a procedure forpurification when necessary. The purification can be carried out byconventional methods for separation and purification of hydrophobicantibiotics. Examples of the methods are column chromatographies orhigh-performance liquid chromatographies, using a column packed with astationary phase such as silica gel, activated alumina, activatedcharcoal, adsorbent resin, etc. The eluent that can be used for silicagel column chromatography includes chloroform, ethyl acetate, methanol,acetone, water, a mixture of thereof, etc.

The resin for high-performance liquid chromatography includeschemically-derivatized silica gel, such as silica gel derivatives havingoctadecyl, octyl, or phenyl groups, and polystyrenic porous polymergels, while the mobile phase that can be used includes aqueous solutionsof water-soluble organic solvents, such as aqueous methanol, aqueousacetonitrile, etc.

TKR2999 of the present invention can each be put to use as such or inthe form of a pharmacologically acceptable salt in medicinalapplications. There is no particular limitation on the type ofpharmacologically acceptable salt. Thus, the salt includes salts ofmineral acids such as hydrochloric acid, sulfuric acid, nitric acid,phosphoric acid, hydrofluoric acid, hydrobromic acid, etc., salts oforganic acids such as formic acid, acetic acid, tartaric acid, lacticacid, citric acid, fumaric acid, maleic acid, succinic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,toluenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid,etc., and salts of alkali metals or alkaline earth metals, such assodium, potassium, calcium, etc.

To administer TKR2999 or its pharmacologically acceptable salt, as adrug, they can be administered to animals inclusive of humans either assuch or in the form of a pharmaceutical composition containing typically0.1 to 99.5%, preferably 0.5 to 90% thereof in a pharmaceuticallyacceptable, non toxic and inert carrier.

The carrier mentioned above includes solid, semisolid or liquiddiluents, fillers, other formulation auxiliaries, etc. and such carrierscan be used alone or in combination.

The above-mentioned pharmaceutical composition is preferablyadministered in unit dosage forms and can be administered orally,parenterally, topically (e.g. transdermally) or rectally. Of course,those pharmaceutical compositions should be administered in dosage formssuited for the respective route of administration.

For administration of TKR2999, or its pharmacologically acceptable salt,as a drug, the dose as an antifungal agent is preferably selected withreference to patient factors such as age and body weight, route ofadministration, nature and severity of disease, etc. Usually in man,however, the daily dose of the active ingredient for an adult patient is10 to 2000 mg. While a daily dose lower than the above range may besufficient in some cases, a dose higher than the range may be requiredin other cases. When a high dose is used, the daily dosage is preferablyadministered in several divided doses.

The above oral administration can be made using solid, powdery, orliquid dosage forms such as bulc powders, powders, tablets, dragees,capsules, drops, subligual tablets, etc.

For the above parenteral administration, liquid unit dosage forms forsubcutaneous, intramuscular, or intravenous administration, typicallysolutions and suspensions, can be employed. These preparations can bemanufactured by suspending or dissolving a predetermined amount ofTKR2999, or a pharmacologically acceptable salt thereof, in a nontoxicliquid carrier suitable for injection, such as an aqueous medium or anoily medium, and sterilizing the resulting suspension or solution.

The topical administration (e.g. transdermal administration) can becarried out using a variety of topical dosage forms such as liquids,creams, powders, pastes, gels, and ointments. These dosage forms can bemanufactured by using a predetermined amount of TKR2999 or apharmacologically acceptable salt thereof, in combination with one ormore of the perfume, coloring agent, filler, surfactant, humectant,emollient, gelatinizer, carrier, preservative, stabilizer, etc.,suitable for topical dosage formulations.

The rectal administration can be made using, for example, suppositorieseach mixing a predetermined amount of TKR2999, or its pharmacologicallyacceptable salt of the present invention, with a low-melting solid basesuch as higher esters, e.g. myristyl palmitate, polyethylene glycol,cacao butter, or a mixture of them.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an ultraviolet absorption spectrum of the antibiotic TKR2999.The ordinate represents absorbance and the abscissa representswavelength (nm).

FIG. 2 is an infrared absorption spectrum of the antibiotic TKR2999. Theordinate represents transmittance (%) and the abscissa represents wavenumber (cm⁻¹).

FIG. 3 is an ¹H-NMR spectrum of the antibiotic TKR2999. The ordinaterepresents the intensity of signal and the abscissa represents chemicalshift (ppm).

FIG. 4 is a ¹³C-NMR spectrum of the antibiotic TKR2999. The ordinaterepresents the intensity of signal and the abscissa represents chemicalshift (ppm).

FIG. 5 shows an HPLC elution pattern of the antibiotic TKR2999. Theabscissa represents the relative intensity of ultraviolet absorption andthe ordinate represents retention time (min.).

BEST MODE OF CARRYING OUT THE INVENTION

A loopful of the TKR2999-strain (FERM BP-6524) from a slant culture wasused to inoculate into a 500-ml Erlenmeyer flask containing 100 ml ofliquid medium (Difco yeast nitrogen base 0.67% (w/v) and glucose 2.0%(w/v)) and incubated on a shaker at 25° C. for 10 days to prepare a seedculture. This seed culture, 1.0 ml, was transferred to each of 26Erlenmeyer flasks of 500 ml capacity each containing 120 ml of the sameliquid medium as above and incubated (under shaking at 220 rpm) at 25°C. for 12 days. The culture broth obtained was centrifuged and separatedinto the supernatant and the cells.

The cells obtained were mixed with 1 L of methanol and subjected tosufficient mixing for extraction. The extract was concentrated underreduced pressure. The residue was mixed with 300 ml each of water andbutanol, and subjected to sufficient mixing to perform extraction withbutanol. The extract was concentrated under reduced pressure to recover362 mg of a residue. The residue was dissolved in 2 ml of methanol andsubjected to high-performance liquid chromatography to provide an activefraction. The fraction was concentrated under reduced pressure torecover 1.2 mg of purified TKR2999 as white powder. The high-performanceliquid chromatography was carried out under the following conditions.

Apparatus: LC-8A (Shimadzu)

Column: YMC pack C18 (2.0 cm×25 cm) (YMC)

Mobile phase: 60% (v/v) of acetonitrile/water containing 0.05%trifluoroacetic acid

Physicochemical Properties

Mass spectrometry was carried out by JMS-DX302 mass spectrometer (JeolLtd.). ¹H-NMR spectrum (in deuterated dimethylsulfoxide with deuterateddimethylsulfoxide as reference) and ¹³C-NMR spectrum (in deuterateddimethylsulfoxide with deuterated dimethylsulfoxide as reference) weremeasured by JNM-A500 nuclear magnetic resonance spectrometer (JeolLtd.). Ultraviolet spectrophotometry (in methanol) was carried out byUV-250 self-recording spectrophotometer (Shimadzu), and infraredabsorption spectrometry (KBr method) was by 270-30 infraredspectrophotometer (Hitachi). Physicochemical properties of the substanceTKR2999 are desribed below.

(1) Mass Spectrometry and Determination of the Molecular Formula

The purified white powdery product available upon vacuum concentrationof the active fraction in said high-performance liquid chromatographywas found to be a substance with m/z 971 [M+H]⁺ by measurement withFAB-MS. In addition, its high resolution FAB-MS was measured and an[M+H]⁺ was observed at m/z 971.5776. This result gave the molecularformula of C₄₄H₇₈N₁₀O₁₄ (calculated, 971.5699) to TKR2999.

(2) Ultraviolet Absorption Spectrum

The UV absorption in methanol of the purified white powdery productavailable upon vacuum concentration of the active fraction in thehigh-performance liquid chromatography was found to be end absorption.

The UV absorption spectrum is shown in FIG. 1.

(3) Infrared Absorption Spectrum

The IR spectrophotometric characterization (KBr method) of the purifiedwhite powdery product available upon vacuum concentration of the activefraction in the high-performance liquid chromatography was as follows.IR (KBr) (cm⁻¹): 3320, 2920, 1680, 1540, 1210, 1140, 840, 800, 720.

The IR absorption spectrum is shown in FIG. 2.

(4) ¹H-NMR and ¹³C-NMR Spectra

The purified white powdery product available upon vacuum concentrationof the active fraction in the high-performance liquid chromatography wasused to measure ¹H NMR spectrum and ¹³C NMR spectrum.

The ¹H-NMR spectrum and ¹³C-NMR spectrum of this product are presentedin FIG. 3 and FIG. 4, respectively.

(5) Amino Acid Analysis

The purified white powdery product available upon vacuum concentrationof the active fraction in the high-performance liquid chromatography washydrolysed in 6 N HCl at 110° C. for 24 hours. The decomposed productwas analyzed by the amino acid analyzer, and aspartic acid, threonine,serine, glycine, and alanine were detected. Additionally β-alanine andornithine were detected.

(6) As for the solubility of the this substance in various solvents, itwas soluble in methanol, but practically insoluble in hexane, chloroformand water.

Based on the above analyses, the purified white powdery productavailable upon vacuum concentration of the active fraction in thehigh-performance liquid chromatography was identified to be TKR2999.

The above TKR2999 was analyzed by reversed-phase partitionhigh-performance liquid chromatography (HPLC) using LC-10Ahigh-performance liquid chromatography (Shimadzu). This HPLC analysiswas carried out under the following conditions.

Column: CAPCELL PAK C₁₈ (6 mm×150 mm) (Shiseido)

Mobile phase: 50% (v/v) acetonitrile/water containing 0.05%trifluoroacetic acid

Column temperature: 40° C.

Detection UV wavelength: 220 nm

As a result, the above TKR2999 was eluted at the position indicated inFIG. 5.

Biological Characteristics

The TKR2999 obtained was tested for the antimicrobial spectrum tovarious microorganisms. Using the liquid medium dilution method for themeasurement, the concentration causing substantially complete inhibitionof micorbial growth was determined as the minimal inhibitoryconcentration (μg/ml). The results are shown in Table 2. The minimalconcentration causing partial inhibition of fungal growth was determinedas the sub-inhibitory concentration (μg/ml) and are shown in parenthesesin the table. In the table, YNBG stands for a medium comprising 0.67% ofyeast nitrogen base (Difco) and 1.0% of glucose, and BHI does for amedium comprising 0.5% of brain heart infusion bouillon (Nissui).

TABLE 2 Minimal inhibitory concentration Test strain Medium (μg/ml)Candida albicans TIMM0136 YNBG 6.25 Candida kefyr TIMM0301 YNBG 12.5(6.25) Cryptococcus neoformans TIMM0354 YNBG 6.25 (3.13) Aspergillusfumigatus TIMM1776 BHI 0.78

It is apparent from Table 2 that TKR2999, the antibiotic according tothe present invention, is active against pathogenic fungi such asCandida albicans, Candida kefyr, Cryptococcus neoformans, Aspergillusfumigatus, etc. Intraperitoneal administration of the TKR2999 obtainedabove at a dose of 50 mg/kg to ICR mice caused no toxic signs.

INDUSTRIAL APPLICABILITY

The present invention provides the antibiotic TKR2999 which are of usein clinical medicine, for example in the therapy of fungal infectiousdiseases, and a method for production of the substance.

What is claimed is:
 1. The antibiotic TKR2999 having the physicochemicalproperties of (1), (2), (3), (4), and (5) described below and itspharmacologically acceptable salt: (1) the mass spectrum by FAB-MSmethod gives m/z 971 as [M+H]⁺; (2) the molecular formula is representedby C₄₄H₇₈N₁₀O₁₄, and its mass spectrum by high-resolution FAB-MS givesm/z 971.5776 as [M+H]⁺; (3) the ultraviolet absorption spectrum inmethanol has an end absorption; (4) the infrared absorption spectrum byKBr method shows the major absorption wave numbers at 3320, 2920, 1680,1540, 1210, 1140, 840, 800, and 720 cm⁻¹; (5) aspartic acid, threonine,serine, glycine, alanine, β-alanine, and ornithine are detected by theamino acid analysis using ninhydrin reaction; and (6) the solubility isthat it is soluble in methanol, and practically insoluble in hexane,chloroform, and water.
 2. A method for producing the antibiotic TKR2999,which comprises cultivating a strain of microorganism belonging to theclass Fungi Imperfecti and capable of producing TKR2999 and harvestingthe objective antibiotic from the resulting culture broth.
 3. Abiologically pure culture of a microorganism belonging to the classFungi Imperfecti which produces the antibiotic TKR2999.